Attenuated recombinant mycobacteria useful as immunogens or as vaccine components

ABSTRACT

This invention provides recombinant mycobacterium strains of pathogenic origin that have been attenuated by the inactivation of a gene coding for a metabolic protein, specifically a gene coding for a protein necessary for the biosynthesis of a purine or a pyrimidine base, and more precisely, the purC gene that codes for an enzyme of the metabolic pathway of purine biosynthesis. The recombinant mycobacterium of this invention have a reduced capacity to propagate in a mammalian host, but remain viable in the host for a period of time sufficient to induce an protective immune response against the natural pathogenic mycobacterium counterpart.

This application claims benefit of provisional application No.60,049,390 filed Jun. 11, 1997.

BACKGROUND OF THE INVENTION

The concept of using attenuated pathogenic bacteria as a vaccinecomponent has been widely disclosed and practiced. The methods forobtaining such attenuated bacteria involve selecting random mutants bychemically- or irradiation- induced mutants or producing recombinantbacteria of pathogenic origin in which a gene involved in some metabolicpathway of the bacteria has been inactivated by genetic engineering.

Straley et al. (1984) have studied the survival of mutant avirulentYersinia pestis that carry defects in one or several metabolic pathways.

Noriega et al. (1994) have genetically engineered an oral Shigellastrain for use as a vaccine prototype by introducing deletions in a gene(aroA) coding for a protein involved in the metabolic pathway of thearomatic amino acids and have demonstrated that the defectiverecombinant resultant Shigella strains were able to induce protectiveantibodies against the wild pathogen.

Substantial work has been also done using Salmonella as a model. See,for example the reports of Hoiseth et al. (1981), Levine et al. (1987),Oyston et al. (1995) and Curtiss (1990).

However, similar work has not yet been done for Mycobacteriumtuberculosis, the etiologic agent of tuberculosis (TB), which infectsone-third of the world's population and kills 3 million people eachyear. TB is the largest cause of death in the world caused by a singleinfectious organism (Bloom and Murray, 1992). According to the WHO, morepeople died from TB in 1995 than in any other year in history.Worldwide, 60 million people suffer from active TB and annually 7million new cases arise (Dolin et al., 1994). It has been estimatedthat, at current rates, up to half a billion people will suffer from TBin the next 50 years.

Efficient chemotherapy exists but requires lengthy and expensivetreatments, making its widespread use and control difficult to achievein developing countries. Prophylactic vaccination against tuberculosiswith the attenuated stain of bovine mycobacteria, BCG (Mycobacteriumbovis Bacillus Calmette—Guérin), is more cost effective and has indeedbeen employed worldwide. Although BCG vaccination has providedprotection against tuberculosis in certain populations, the variation inefficacy of this vaccine in different field trials and its modestprotective effect against the adult form of the disease (estimated bymeta-analysis to be about 50%) (Colditz et al., 1994) are points ofmajor concern. These considerations have led the WHO to place TB controlefforts, notably through the development of new vaccines, among its toppriorities.

However, despite its importance, the genetic determinants of M.tuberculosis virulence remain poorly characterized. In the recent years,considerable efforts have been made towards the identification ofindividual mycobacterial antigens involved in the immune response totuberculosis (Young et al., 1992) with the aim of developing subunitvaccines. The observation that only live vaccines confer high levels ofprotective immunity against tuberculosis (Weiss & Dubos, 1955; Orme,1988), in addition to the fact M. tuberculosis short-term culturefiltrates containing proteins secreted by actively replicating bacteriawere shown to protect mice against a subsequent challenge with thevirulent strain (Andersen, 1994), suggested that proteins secreted by M.tuberculosis might be good candidates for the design of subunitvaccines. Indeed, antigens such as ESAT 6 (Andersen et al., 1995), mpt64(Haslov et al., 1995), the antigen from the 45/47 kDa complex (Romain etal., 1993) and the components of the antigen 85 complex (Wiker & Harboe,1992) were identified as powerful immunogens eliciting delayed-typehypersensitivity (Haslov et al., 1995; Romain et al., 1993), antibodyresponses (Romain et al., 1993; Wiker & Harboe, 1992) and theproliferation of T-lymphocyte populations responsible for long-livedimmunity (Andersen et al., 1995) in guinea pigs or mice. Some of theseantigens showed protective efficacy in the mouse model of tuberculosiswhen used as DNA vaccines (Huygen et al., 1996).

An alternative strategy to develop novel vaccines consists ofconstructing mutant strains of mycobacteria that are rationallyattenuated. The recent development of genetic tools for performingsite-specific (Pelicic et al., 1997) and random-site mutagenesis(Pelicic et al., 1997; Bardarov et al., 1997) in organisms from theMycobacterium tuberculosis complex now renders feasible theaccomplishment of such a goal. Live vaccines should have advantages oversubunit vaccines in that i) they represent a greater pool of antigenswhich presumably should cover a wider range of T-cell repertoires, andii) they are generally more cost effective to produce. Moreover,attenuated mutants of M. tuberculosis should express homologousprotective antigens which the BCG strains lack, and, thus, elicit a morespecific and stronger protective immune response against virulencechallenge. In support of this hypothesis, the molecular analysis byMahairas and collaborators (1996) of genetic differences between M.bovis BCG and its virulent counterparts M. bovis and M. tuberculosisclearly established the existence of regions of deletion in the genomeof BCG (representing about 30 kb in all), some of which contain the ORFsencoding the highly immunogenic ESAT 6 and mpt64 antigens (the latterbeing absent from certain BCG strains only (Oettinger & Andersen,1994)).

Among attenuated strains of intracellular bacterial pathogens,auxotrophic mutants carrying defects in the shikimate or the purinebiosynthetic pathways were shown to be of particular interest aspotential live vaccines candidates because they are attenuated in vivoand have the ability to retain their immunogenicity. Some Salmonella,Yersinia and Corynebacteria purine and aromatic amino acid auxotrophshave LD50s in mice 3 to 6 log₁₀ higher than that of the wild type(Hoiseth & Stocker, 1981; O'Callaghan et al., 1988; McFarland & Stocker,1987; Bowe et al., 1989; Simmons et al., 1997). These Salmonellaauxotrophs as well as a Brucella purE deficient mutant are, however,able to persist several weeks in mice (Crawford et al., 1996;O'Callaghan et al., 1988) and the aroA and purA mutants of Salmonellatyphimurium are able to induce protective immunity in mice against achallenge with the virulent strain (Hoiseth & Stocker, 1981; McFarland &Stocker, 1987).

However, the extreme difficulty in creating defined mutants of M.tuberculosis, either by allelic exchange or transposon mutagenesis, hasprevented identification of its virulence factors following Koch'spostulates (Falkow, 1988; Jacobs, 1992). Rather, alternative geneticstrategies have been used, including complementation of non-pathogenicbacteria (Arruda et al, 1993) and spontaneous avirulent mutants withlibraries of virulent M. tuberculosis (Pascopella et al. 1994) or M.bovis (Collins et al., 1995) chromosomal DNA. Although these studieshave identified genes required for entry into epithelial cells andconferring a growth advantage in vivo, the great majority of themycobacterial genes involved in virulence remain unknown. Developingefficient mutagenesis systems is thus a top priority for mycobacterialgenetics.

One method for creating mutants is allelic exchange mutagenesis.Recently, low-frequency allelic exchange was demonstrated in bacteria ofthe M. tuberculosis complex using a suicide delivery vector (Reyrat etal., 1995; Azad et al., 1996), and new protocols allowing easierdetection of allelic exchange mutants have also been developed Norman etal., 1995; Balasubramamian et al., 1996; Pelicic et al., FEMS Microbiol.Lett. 1996). However, detection of very rare allelic exchange events ishindered by low transformation efficiencies and high frequencies ofillegitimate recombination. Thus, many mycobacterial genes still remainrefractory to allelic exchange by available technology.

Clearly, the allelic exchange mutagenesis system requires the design ofmore efficient methods. The problems encountered can be circumvented byusing a replicative delivery vector which is efficiently lost undercertain conditions. Allowing the introduced delivery vector to replicateavoids the problems arising from low transformation efficiencies. Then,under counter-selective conditions, clones that still contain the vectorare eliminated, allowing the detection of very rare genetic events. Onesuch system has recently been developed. Using a conditionallyreplicative vector which is efficiently lost at 39° C. in M. smegmatis,the first mycobacterial insertional mutant libraries were constructed inthis fast-growing model strain (Guilhot et al., 1994). However, thethermosensitive vectors used are only weakly thermosensitive inslow-growing mycobacteria of the M. tuberculosis complex and thereforecannot be used in these species for allelic exchange mutagenesis(unpublished data).

To date, it has not been possible to inactivate any specific gene of amycobacterium strain via allelic exchange due to the absence of anefficient positive counter-selective marker gene that allows forselection of recombinant mycobacteria carrying a defective metabolicpathway gene. Thus, it has not previously been possible to generate amycobacterium strain with an inactivated gene, particularly a geneinvolved in a metabolic pathway of the pathogenic mycobacteria, suchthat the defective strain is able to replicate only at a very low levelin the host but does persist in the host long enough to allow theinduction of an immune response. Nor has it been possible to produce anattenuated recombinant mycobacterium strain that is incapable ofinducing disease in a host to which it has been administered.

SUMMARY OF THE INVENTION

This invention provides for the first time, recombinant mycobacteriumstrains of pathogenic origin that have been attenuated by theinactivation of a gene coding for a metabolic protein, specifically agene coding for a protein necessary for the biosynthesis of a purine ora pyrimidine base, and more precisely, the purC gene that codes for anenzyme of the metabolic pathway of purine biosynthesis.

Construction of recombinant mutant auxotrophs of Mycobacterium via anallelic exchange event has allowed the isolation of new live attenuatedstrains of M. tuberculosis of immunogenic and/or vaccinal value. Usingthe purC gene from M. tuberculosis (Jackson et al., 1996), auxotrophicmutants for the purine bases of M. bovis-BCG (vaccinal Pasteur strainnumber 1173P2, which is publicly available at the Pasteur InstituteCollection) and of M. tuberculosis (clinical isolate Mt103) have beenconstructed via an allelic exchange event.

Thus, it is an object of this invention to provide recombinantmycobacterium of pathogenic origin that have a lower capacity topropagate in a mammalian host, specifically a human, but which remainviable in the host for a period of time sufficient to induce an immuneresponse, and preferably, a protective immune response, against thenatural pathogenic mycobacterium counterpart.

It is a further object of this invention to provide attenuatedrecombinant mycobacterium incapable of inducing a disease in a host towhich they have been administered.

Mycobacterium species, as described herein, may be any strain of theMycobacterium genus, including, but not limited to: Mycobacteriumtuberculosis complex such as M. Bovis-BCG, M. bovis, M. tuberculosis, M.africanum and M. microti; M. avium; M. intracellulare; and Mycobacteriumleprae.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a map of plasmid p27CKX.

FIG. 2 depicts the design of new vectors for positive selection of raregenetic events (pPR27 is shown as an example). Only single restrictionsites which can be used for the subsequent cloning of a transposon or amutant allele are shown.

FIG. 3 depicts a Southern-blot analysis of M. tuberculosis purC mutantsand the expected schematic pattern of hybridization for an allelicexchange mutant. Five auxotrophic mutants were picked at random (clones1 to 5). Chromosomal DNA was digested with BamHI and probed forhybridization with the p27CKX vector. M. tuberculosis DNA was includedas a control (WT). Molecular weights are indicated in kb.

FIG. 4 depicts the number of colony forming units found in macrophagesinfected in vitro with either wild type M. bovis BCG (Pasteur strain1173P2) (-□-) or the corresponding recombinant mutant strain MYC 1552 (. . . ⋄ . . . ).

FIG. 5 depicts the number of colony forming units found in macrophagesinfected in vitro with wild type M. tuberculosis Mt103 (-□-), thecorresponding recombinant mutant strain, MYC 1551 ( . . . ∘ . . . ) andthe recombinant mutant strain complemented with vector carrying the purCgene ( . . . ⋄ . . . ). These results demonstrate that the MYC-1551strain is not replicating due to the absence of a functioning purC gene.

FIG. 6 depicts the number of colony forming units found in variousorgans of mice infected in vivo with wild type M. bovis BCG (Pasteurstrain 1173P2) ( . . .  . . . ), recombinant mutant strain MYC 1552plated and cultivated (after sacrifice of the infected animal) inhypoxanthin ( . . . ∘ . . . ) and recombinant mutant strain MYC 1552plated and cultivated in hypoxanthin and kanamycin (-□-). These resultsdemonstrate that the mutant strain is maintained in the infected organbut is not replicating.

FIG. 7 depicts the number of colony forming units found in the lungs ofmice infected in vivo with wild type M. tuberculosis Mt103 (FIG. 7A) andwith the corresponding recombinant mutant strain, MYC 1551 (FIG. 7B).

FIG. 8 depicts the number of colony forming units found in the liver ofmice infected in vivo with wild type M. tuberculosis Mt103 (FIG. 8A) andwith the corresponding recombinant mutant strain, MYC 1551 (FIG. 8B).

FIG. 9 depicts the number of colony forming units found in the spleen ofmice infected in vivo with wild type M. tuberculosis Mt103 (FIG. 9A) andwith the corresponding recombinant mutant strain, MYC 1551 (FIG. 9B).

FIG. 10 depicts the persistence of MYC1551 and MYC1552 withinbone-marrow macrophages. In this representative experiment, 5×10⁴macrophages were infected with 5×10⁴ viable bacilli and the growth wasmeasured over time. M. bovis BCG 1173 P2 (-□-), or the BCG auxotroph(MYC1552) ( . . . ⋄ . . . ) were used to infect the macrophages (FIG.10A). A M. tuberculosis virulent strain (MT103) (-□-), its auxotrophcounterpart (MYC1551) ( . . . ⋄ . . . ) or MYC1551 harbouring theplasmid pMJ105 (carrying the purC gene) ( . . . ∘ . . . ) were used toinfect the macrophages (FIG. 10B). Each point represents the geometricmean +/− the standard deviation (SD) of two independant cultures.

FIG. 11 depicts persistence of M. tuberculosis MT103 and the purineauxotroph MYC1551 in mice. BALB/c mice were infected intravenously with10⁷ viable units of either a M. tuberculosis virulent strain (MT103)(-□-) or MYC1551 ( . . . ⋄ . . . ), and the persistence of bacteria inthree organs, spleen (FIG. 11A), liver (FIG. 11B), and lung (FIG. 11C)were measured over time. The value indicated represents the geometricmean+SD obtained with five different mice.

FIG. 12 depicts persistence of M. bovis BCG and its auxotrophcounterpart, MYC1552, in mice. BALB/c mice were infected intravenouslywith 10⁷ viable units of either M. bovis BCG (-□-) or MYC1552 ( . . . ⋄. . . ), and the persistence of bacteria in three organs, spleen (FIG.12A), liver (FIG. 12B), and lung (FIG. 12C) were measured over time. Thevalue indicated represents the geometric mean+SD obtained with fivedifferent mice.

DETAILED DESCRIPTION

A better understanding of Mycobacterium tuberculosis virulencemechanisms is highly dependent on the design of efficient mutagenesissystems. A system enabling the positive selection of insertional mutantshaving lost the delivery vector has now been developed. This system isefficient for gene exchange mutagenesis and has been demonstrated withthe purC gene: 100% of the selected clones were allelic exchangemutants. Therefore, a single, simple methodology has enabled thedevelopment of powerful mutagenesis systems, the lack of which haspreviously been a major obstacle to the genetic characterization of M.tuberculosis.

Thus, using the allelic exchange method of Pelicic et al. (Mol.Microbiol., 1996) with an interrupted purC gene of Mycobacteriumtuberculosis, it has been surprisingly discovered that the inactivationof the purC gene leads to recombinant M. tuberculosis strains that arestill able to persist in a macrophage of a host, but have totally orpartially lost their ability to replicate and lyse the macrophage incomparison with the corresponding wild strain. This surprising discoveryhas now enabled the design of new mycobacterial strains of pathogenicorigin suitable as vaccine components.

The vectors used in this invention to select recombinant mycobacteriaclones that have undergone an allelic exchange at the location of thegene of interest (i.e., the gene to be inactivated) are principallycharacterized by the following features:

a) the vector carries a conditional lethal counter-selective markergene, for example the SacB gene, such as described in Pelicic et al.,Mol. Microbiol. 1996;

b) the vector carries, preferably in the neighborhood of the conditionallethal counter-selection marker, an inactivated copy of the gene ofinterest (for example an interrupted gene involved in the biosynthesisof purine or pyrimidine bases, such as purC gene) that will replace itswild counterpart in the mycobacterial genome (chromosome or plasmid);

c) the vector optionally carries a conditionally functional origin ofreplication, such as a thermosensitive (ts) origin of replication whichallows the control, during the allelic exchange method processing, ofthe vector replication; and

d) the vector optionally carries an additional selection marker gene,preferably, an antibiotic resistance marker gene such as, for example,gentamycin, hygzomycin, or kanamycin resistance which may be used forpre-selecting the mycobacteria that have received the recombinantvector, whether they have undergone an allelic exchange event or not.

In the most preferred embodiment, this additional marker gene isinserted in the gene of interest in order to inactivate said gene ofinterest. FIG. 1 describes a preferred vector according to thisinvention, plasmid p27CKX.

In another preferred embodiment, the vector of this invention containsthe SacB gene and a nucleotide sequence comprising a gene coding for aprotein necessary for the biosynthesis of a purine or a pyrimidine basethat has been inactivated by addition, deletion or substitution of atleast one base pair and preferably from 10 to 20 base pairs. Preferably,the gene to be inactivated is a gene coding for guanine or adenine.Alternatively, the gene to be inactivated is a gene coding for a baseselected from the group consisting of cytosine, uracil or thymine. In amore preferred embodiment, the gene to be inactivated is the purC geneor the purL gene.

In one embodiment of this invention, recombinant mutant mycobacteria,and specifically the purC mutant, of M. tuberculosis have beenconstructed following a selection protocol based on the use of thecounter-selective marker gene SacB and of a thermosensitive conditionalreplication vector according to this invention.

In an optional pre-selection step of the selection protocol,mycobacteria that have received the recombinant vector are selected on aculture medium supplemented with an appropriate molecule, such as thecorresponding antibiotic if the vector contains an antibiotic resistancemarker gene.

Then, the mycobacteria or the pre-selected mycobacteria, are propagatedat a permissive temperature in order to establish a high copy number(e.g., 3 to 5 copies) of the recombinant vector in every bacterium andgenerate a large number of bacteria carrying the recombinant vector.These conditions will greatly enhance the probability of an allelicexchange event occurring. In optimal conditions, the transfectedmycobacteria may carry up to five recombinant vectors per bacteriumcell.

Next, the mycobacteria are incubated in a medium supplemented withsucrose and at a non permissive temperature (to stop the recombinantvector replication). Because the mycobacteria are still multiplying whenplaced at a non-permissive temperature, there is a dilution of therecombinant vector occurrence in the bacteria. Selecting bacteria thatcarry the antibiotic resistance gene will provide mainly mycobacteriathat have the recombinant vector material inserted in their genome,either after an allelic exchange event, by a single cross-over in thepurC locus, or by the random integration of the plasmid cassette in themycobacterial genome. Simultaneously selecting bacteria that stillpropagate in a culture medium supplemented with sucrose will ensure thatonly the recombinant mycobacteria that have undergone an allelicexchange event and have lost the recombinant vector parts that carry theSacB gene survive. Thus, the combination of the ts gene action and theSacB gene action acts as a synergistic mechanism that allows selectionof allelic exchange recombinant mycobacteria at a high rate.

The resultant recombinant mycobacteria positively selected thus combinethe features of having the recombinant gene of interest integrated intheir genome but having lost the whole remaining recombinant vectorsequences. These bacteria are then necessarily those which haveundergone an allelic exchange event. Specifically, the purC recombinantauxotrophic mutant of M. tuberculosis has been constructed using plasmidp27CKX. A map of plasmid p27CKX is shown in detail in FIG. 1.

At the end of the different selection steps, 100% of the selected cfu(colony forming units) correspond to purC mutants of M. tuberculosis.This result has been confirmed by Southern blotting of 17 cfu (data notshown) and also by phenotypic assays (with minimum culture medium Sautonsupplemented with hypoxanthin) on 96 cfu.

The intramacrophagic survival of the purC mutant of M. tuberculosis hasbeen compared with the corresponding wild strain Mt103. Primary culturesof bone marrow macrophages from Balb/c mice have been infected with thewild or the recombinant auxotrophic strains of Mt103. The infectionmultiplicity was 40 for Mt103. At day 1, no difference was observedbetween the wild and the recombinant auxotrophic strains. At day 4, itwas estimated that macrophages were infected by tenfold more with wildstrains than with the auxotrophic recombinant strains. This difference,both in the proportion of infected macrophages and in the number ofbacteria per macrophage cell increases with time. At day 8, the wildstrain Mt103 had lysed almost all the cultured macrophages while for therecombinant auxotrophic strain Mt103 purC, the cell layer was notaffected, with only 10 to 20% infected macrophages and 5 to 10 bacteriaper cell. Thus, the auxotrophic recombinant strains have anintramacrophagic multiplication considerably decreased in comparisonwith the corresponding wild strains.

Accordingly, this invention pertains to a recombinant mycobacteriumstrain of pathogenic origin capable of replicating in the macrophage ofa host, said strain containing in its chromosome or on a natural plasmida gene coding for a protein necessary for the biosynthesis of a purineor a pyrimidine base that is inactivated by at least one point mutationor by addition, deletion or substitution of at least one base pair andpreferably from 10 to 20 base pairs. An important feature of thisinvention is that the alteration of the sequence of the inactivated geneof interest must be sufficient in order to avoid reversion.Consequently, a preferred embodiment of the inactivated gene of thepresent invention is that it is interrupted by an exogenouspolynucleotide.

In a specific embodiment of the present invention, the exogenouspolynucleotide comprises an antibiotic resistance gene that is used in apre-selection step in the selection protocol used to obtain therecombinant attenuated strains. In another embodiment, the exogenouspolynucleotide sequence used to interrupt the gene of interest comprisesa gene coding for an antigenic protein heterologous with respect to themycobacterium stain to be transfected. In another specific embodiment ofthis invention, the recombinant mycobacteria of the invention aretransformed with an expression vector carrying a polynucleotide encodingan antigenic peptide (i.e., a polynucleotide encoding at least oneantigenic epitope) which is heterologous with respect to the strain tobe transfected. A preferred recombinant vector is, for example, a vectorof the PAL 5000 family. These embodiments may be particularly useful inthe design of new vaccines against unrelated pathogens. Illustrativeembodiments of antigenic peptide or protein encoding sequences aredescribed below.

Illustrative embodiments of the heterologous antigenic protein to beexpressed by recombinant mycobacteria of the present invention include:

1) the nucleotide sequence coding for the whole desaturase antigen of M.tuberculosis or at least an antigenic portion of the desaturase antigen.(For example, desaturase derived nucleotide sequence may be inserted inthe M. bovis BCG stain as a fusion sequence with the gene of interestthat codes for a protein involved in the biosynthesis of the purine orpyrimidine bases. This represents an improvement to the conventionallyused BCG strains as such recombinant strains will be able to expressimmunogenic determinants that are specific of M. tuberculosis. Suchstrains should provide improved means to make efficient vaccinepreparations.);

2) the 45/47 kD immunogenic protein from M. tuberculosis described inPCT application number PCT/FR 96/0166;

3) the surface antigen from the Hepatitis B virus (HBsAg) described inthe French patent application number FR 7921811;

4) the nucleotide sequences coding for all or part of HIV glycoproteins,e.g., the genome sequences from HIV-1 described in patent applicationsGB 8324800, EP 84401834 or EP 85905513 and the genome sequences forHIV-2 described in the patent application EP 87400151;

5) the nucleotide sequences coding for the 65 kD antigen of M.tuberculosis (Huygen et al., 1996); and

6) the nucleotide sequence coding for human tumor antigens, such as MAGEantigen, and specifically MAGE-3 antigen, such as described in the U.S.Pat. No. 5,591,430.

It will be appreciated that this invention additionally encompassesimmunogenic compositions comprising recombinant mycobacterium strainsdescribed above. The invention also encompasses a vaccine compositioncontaining a recombinant mycobacterium according to this invention incombination with a pharmaceutically compatible excipient The presentinvention also pertains to a vaccine composition for immunizing humansand mammals against a pathogenic strain of mycobacteria, comprising animmunogenic composition as described above in combination with apharmaceutically compatible excipient (such as, for example, salinebuffer), and optionally in combination with at least one adjuvant suchas aluminum hydroxide or a compound belonging to the muramyl peptidefamily.

Various methods for achieving adjuvant effect for the vaccine includethe use of agents such as aluminum hydroxide or phosphate (alum),commonly used as 0.05 to 0.1 percent solution in phosphate bufferedsaline, admixture with synthetic polymers of sugars (Carbopol) used as0.25% solution. Another suitable adjuvant compounds consist in DDA(dimethyldioctadecyl-ammonium bromide), as well as immune modulatingsubstances, such as lymphokines (e.g., IFN-gamma, IL-1, IL-2 and IL-12)or IFN-gamma inducers compounds, such as poly I:C.

The vaccine composition according to the present invention isadvantageously prepared as an injectable form (either as liquid solutionor suspension). However, solid forms suitable for solution in orsuspension in, liquid prior injection may also be prepared.

In addition, if desired, the vaccine composition may contain minoramounts of auxiliary substances such as wetting or emulsifying agents,pH buffering agents, or adjuvants which enhance the effectiveness of thevaccines.

The vaccine compositions of the invention are administered in a mannercompatible with the dosage formulation, and in such amount as will betherapeutically effective and immunogenic. The quantity to beadministered depends on the subject to be treated including, e.g., thecapacity of the individual's immune system to induce an immune response.

Suitable dosage ranges are of the order of 10⁴ to 10⁶ cfu (colonyforming units) at an attenuated recombinant mycobacteria concentrationof about 10⁶ cfu/mg. Most preferably, the effective dose is about 10⁵cfu.

The dosage of the vaccine will depend on the route of administration andwill vary according to the age of the patient to be vaccinated and, to alesser degree, the size of the person to be vaccinated. Most preferably,the vaccine composition according to the present invention isadministered via an intradermal route and in a single boost.

In the case of patients affected with immunological disorders such as,for example, immunodepressed patients, each injected dose preferablycontains half the weight quantity of the attenuated mycobacteriacontained in a dose for a healthy patient.

In the case of neonates, the dose will be approximately four times lessthan for an adult, and in the case of young children (4-6 years old),the dose will be approximately half the dose used for an adult healthypatient.

In some instances, it will be necessary to proceed with multipleadministrations of the vaccine composition according to the presentinvention, usually not exceeding six administrations, more usually notexceeding four vaccinations, and preferably one or more, usually atleast about three administrations. The administrations will normally beat from two to twelve week intervals, more usually from three to fiveweek intervals. Periodic boosters at intervals of 1-5 years, usuallythree years, will be desirable to maintain the desired levels ofprotective immunity.

The invention further encompasses polynucleotides containing all or partof the genome of a mycobacterium strain which is devoid of a wild geneencoding a protein involved in the biosynthesis of a purine or apyrimidine base.

Representative embodiments of this invention will be described in moredetail in the following examples.

EXAMPLE 1

Bacterial Strains and Culture Conditions

E. coli strain DH5α was used in this study for cloning experiments, andwas grown on liquid or solid Luria-Bertani (L) medium. M. smegmatismc²155 (Snapper et al., 1990), M. tuberculosis 103 (isolated from a TBpatient) and M. bovis BCG strain Pasteur 11732P were grown on liquidMiddlebrook 7H9 medium (Difco) supplemented with 0.2% glycerol and 0.05%Tween, or on solid Middlebrook 7H10 medium (Difco). When required,antibiotics were included at the following concentrations: kanamycin (20μg/ml) and gentamycin (5 μg/l) for mycobacteria, and gentamycin (20μg/ml) for E. coli. Where indicated, 10% or 2% sucrose was added for M.smegmatis or bacteria of the M. tuberculosis complex respectively(Pelicic et al., FEMS Microbiol Lett. 1996; Pelicic et al., J.Bacteriol. 1996).

Purine auxotrophs were identified by their inability to grow on Sautonmedium, unless the medium was supplemented with hypoxanthin (20 μg/ml).Briefly, single colonies were picked and resuspended in 96-wellmicroliter plates containing Sauton medium with or without hypoxanthinsupplement. The plates were incubated at 37° C. under 5% CO₂. Growth wasestimated by following the opacity in adjacent wells with and withouthypoxanthin addition.

Electrotransformation

Electrocompetent cells were prepared as previously described (Pelicic etal., Mol. Microbiol. 1996) with minor modifications. M. tuberculosis andM. bovis BCG were grown in 200 ml of 7H9 medium to an OD₆₀₀ of 0.4.Cells were washed three times in 10% glycerol and resuspended in 1 ml10% glycerol. Aliquots (100 μl) of freshly prepared competent cells wereelectroporated in the presence of 1 μg of vector DNA in 0.2 cm cuvettes(Biorad) with a single pulse (2.5 kV; 25 pF; 200 ohms). Five ml of freshmedium was then added and the culture was incubated at 32° C. for 24hours before plating, to allow antibiotic resistance expression.Transfomants were scored after 7-8 weeks of incubation at 32° C.

DNA Extraction and Southern Analysis

Mycobacterial genomic DNA was isolated as previously described (Pelicicet al., Mol. Microbiol. 1996) with minor modifications. One hundred μlof D-cycloserine (1 mg.ml⁻¹) was added to a 10 ml saturated culturewhich was then incubated overnight at 37° C. Cells were pelleted bycentrifugation (15 min, 5000×g). The pellet was resuspended in 250 μl ofsolution I (25% sucrose; 50 mM Tris—HCl pH 8.0; 50 mM EDTA; 500 μl.ml⁻¹lysozyme) and incubated overnight at 37° C. Two hundred and fifty μl ofsolution II (100 mM Tris—HCI pH 8.0; 1% SDS; 400 μg.ml⁻¹ Proteinase K)was then added and the samples incubated for 4h at 55° C. The lysate wasthen extracted twice with phenol-chloroform and the DNA was concentratedby ethanol precipitation. Approximately one microgram of genomic DNA wasdigested overnight with an excess of restriction enzyme (30 U) and thefragments separated by electrophoresis through 0.7% agarose gels.Southern-blotting was carried out in 20×SSPE (150 mM NaCl; 8.8 mMNaH₂PO₄; 1 mM EDTA pH 7.4) using Hybond−N+ nylon membranes (Amersham).The Megaprime random-primed labeling kit (Amersham) and 5 μCi of (α-³²p)dCTP were used to label probes. Nonincorporated label was removed byfiltration through a Nick Column (Pharmacia). Prehybridization andhybridization were carried out at 65° C. using RH buffer (Amersham) asrecommended by the manufacturer. Serial 15 min washes were performed at65° C. as follows: two washes with (2×SSPE; SDS 0.1%), one wash with(1×SSPE; SDS 0.1%) and two washes with (0.7×SSPE; SDS 0.1%). BioMax MSX-ray film (Kodak) was exposed for 4h to the blots at −80° C.

Construction of Vectors

The thermosensitive origin of replication of pAL5000, present in ts—SacBdelivery vectors, was extracted from pB4D* on a 5 kb BamHI (wholepAL5000) or a 3.7 kb EcoRV+KpnI (minimal origin of replication) fragment(Guilhot et al., 1992). The fragments were blunt-ended and cloned intoBamI-cut pJQ200 harboring the SacB gene (Quandt and Hynes, 1993). Bothorientations were obtained for the 3.7 kb “short” insert (pPR23-1 andpPR23-2) and only one orientation for the 5 kb insert (pPR27).

Plasmids pPR23 and pPR27 were deposited under the provisions of theBudapest Treaty at the National Collection of Cultures of Microorganisms(C.N.C.M.) in Paris on Jun. 19, 1996 and assigned reference Nos. I-1726and I-1730, respectively.

The purC gene was excised from pMJ1 (Jackson et al., 1996) on a 2.5 kbBamHI fragment and inserted into BamHI-cut pACYC184. The resultingvector was named pMJ100. The aph cassette from pUC4K conferringkanamycin resistance, present on a 1.2 kb PstI fragment, was cloned intopMJ100 at the single PstI site present in purC. PurC::Km was extractedfrom the resulting pMJ101 vector on a 3.7 kb BamHI fragment, blunt-endedand ligated into BamHI-cut pXYL4, an E. coli vector containing xylEbracketed by two BamHI sites. The resulting vector was named pMJ102.p27CKX, the construct used for the allelic exchange, was obtained bytransferring a 4.7 kb BamHI fragment from pMJ102, containing purC::Kmand xylE, into BamHI-cut pPR27.

Multiplication and Persistence of M. bovis-bcg and M. tuberculosis inVarious Organs of Mice

Infections

30 female C57BL6 mice (assay of M. tuberculosis) or Balb/c mice (assayfor M. bovis BCG) were infected with the wild bacteria (Mt103 or Pasteurstrain BCG 1173P2) and 30 mice were infected with the correspondingauxotrophic strain MYC 1551 or MYC 1552. (Recombinant mycobacteriumstrains MYC 1551 and MYC 1552 were deposited under the provisions of theBudapest Treaty at the National Collection of Cultures of Microorganisms(C.N.C.M.) in Paris on May 12, 1997 and assigned reference Nos. I-1871and I-1872, respectively.)

Mice were infected via an i.v. route with 10⁵ cfu of Mt103 or MYC 1551or with 10⁶ cfu of BCG 1173P2 or MYC 1552 that were resuspended in 0.5ml of PBS buffer supplemented with 0.01% tween.

Multiplication assay

The numeration of colony forming units was performed in the threefollowing target organs: spleen, liver and lung.

The assay was performed at days 1, 7, 14, 21, 42 post-infection with M.tuberculosis and at days 1, 7, 14, 28, 42 and 70 post-infection withBCG. For each measurement and each strain, 5 mice were used.

Measurement of the cfu numbers in organs

Organs were recovered and torn to pieces between two glass slides forspleen and lungs or cut then ground with a Stomacher apparatus (2 min.at maximum speed) for the livers. The ground organs were thenresuspended in 10 ml solution I (Sauton diluted to ¼ in sterile waterthen buffered to pH 7.5+OADC (Bacto Middlebrook OADC enrichment Difco)2%+(gentamycin 10 μg/ml). Various dilutions (1, 10⁻¹, 10⁻², 10⁻³, 10⁻⁴)of these preparations were prepared in solution II (Sauton diluted to ¼in sterile water and then buffered at pH 7.5) and spread on Petri dishes7H11 for the wild strains or (7H11+hypoxanthin (20 μg/ml)) or(7H11+hypoxanthin (20 μg/ml)+kanamycin (20 μg/ml)) for the strains MYC1551 and MYC 1552 in order to evaluate the number of cfu per organ.

Infection Protocol or Macrophages by the Strains Mt103, BCG 1173P2,MYC1551 and MYC1552

Preparation of Balb/C mice bone marrow macrophages

A 6-8 week old female Balb/C mouse was killed and the femurs wererecovered. Bone marrow was extracted from the femurs by repeatedpassages of a HBSS solution (Gibco BRL) in the medulla channel with asyringe. Once large particles were sedimented, bone marrow cells wererecovered by centrifugation (1300 rpm during 5 min) and resuspended inMEM modified Dulbecco culture medium (Gibco BRL)+10% fetal calfserum+glutamine+10% L cells supernatant culture medium (containinggrowth factors for macrophages).

Then 5×10⁴ macrophages/well were plated in Lab Tec slides (8 wells) in avolume of 400 μl.

Macrophages infection by the bacteria 8 to 10 days post-isolation

Macrophages in each well of the Lab Tec slides were counted. Bacteriawere thawed (1 ml), washed twice in macrophage culture medium and thenbriefly sonicated in order to disperse them (Ultra-sonicater bath Braun2200 during 15 sec). The bacterial preparations were then put tosediment during 10 min. About 500 μl of the resultant supernatant mediumwere used to perform the infection assays. Various dilutions of thissupernatant medium were spread on Petri dishes 7H10 in order to evaluateprecisely the cfu number that are used to infect macrophages.

The macrophages were then infected with the wild bacteria and therecombinant bacteria. The multiplicity of infection (MOI) is from 0.2 to1 (i.e., 5×10⁴ bacteria per well). The infection was maintained during18h.

Count of the bacteria

The cfu counts perwell was performed at time t=18h, day 4, day 8, day15. At t=18h: the infection was stopped by three washings of the Lab Tecslides in a HBSS medium (removing of the non-internalized bacteria). Ateach subsequent time interval, a count of the cfu in each well wasperformed (four wells/bacterial strain/time point) : macrophages werelysed in a 100 μl lysis buffer volume and various dilutions of thissolution were spread for counting the cfu on Petri dishes 7H10 for thewild strains or (7H10+hypoxantbin 20 μg/ml+kanamycin 20 μg/ml) for thestrains MYC1551 and MYC1552.

For each time point, a Kinyoun straining was performed on the infectedslides, in parallel with the cfu counts, in order to evaluate themacrophage state and to assess in eventual cell lysis.

Design and Testing of a Novel Methodology for the Selection ofInsertional Mutants

Recently, it was demonstrated that expression of the SacB gene from B.subtilis is lethal to mycobacteria in the presence of sucrose. SacB cantherefore be used as a counter-selectable marker (Pelicic et al., FEMSMicrobiol. Lett. 1996; Pelicic et al., J. Bacteriol. 1996; Pelicic etal., Mol. Microbiol. 1996). As described above, whether SacB could beused for the positive selection of insertional mutants was tested. Aseries of conditionally replicative vectors, combining thecounter-selective properties of the SacB gene and a mycobacterialthermosensitive origin of replication were constructed (FIG. 2). Thesets—SacB vectors were introduced into M. smegmatis mc²155 byelectroporation (Snapper et al, 1990). M. smegmatis transformants,selected at 32° C. on 7H10-gentamycin, were grown in 7H9 at 32° C. untilsaturation. The efficiency of the different counter-selections were thenestimated by plating 100 μl samples of these cultures at differenttemperatures on 7H10-gentamycin plates with or without 10% sucrose, andcounting colony-forming units (CFU). Stability of the pAL5000thermosensitive origin of replication was measured by plating samples at39° C., the restrictive temperature for replication, without sucroseaddition. The efficiency of SacB counter-selection was estimated byplating samples at 32° C. in the presence of 10% sucrose. By plating onsucrose plates at 39° C., the global counter-selection was assessed(Table 1). Each of the counter-selective pressures, sucrose and growthtemperature, was individually low and led to only a limited loss of thevector. However, when transformants were counter-selected for both SacBand the thermosensitive origin of replication, the efficiency ofcounter-selection was extremely high (Table 1).

TABLE 1 Effect of sucrose and temperature on growth of M. smegmatistransformed with pPR27.^(a) Growth conditions CFU per 100 μlCounter-selection efficiency 32° C. 1.2 × 10⁷ — 39° C. 5000 4.2 × 10⁻⁴sucrose at 32° C. 3000 2.5 × 10⁻⁴ sucrose at 39° C. 6   5 × 10⁻⁷^(a)Identical results were obtained for pPR23 (data not shown).

The results suggested that ts—SacB vectors could be used to deliver atransposon or a mutated allele into the chromosome of M. tuberculosis,allowing the construction of insertional mutant libraries or geneexchange mutants respectively. This protocol of selection was used forall subsequent mutagenesis experiments. Because trasformants were grownunder permissive conditions, problems due to low transformationefficiencies were avoided. During this step of replication, mutantsarising by allelic exchange or trasposition can accumulate, overcomingproblems due to low frequencies of allelic exchange and transposition.Finally, the great majority of the clones that still contain the vectorwere eliminated, strongly increasing the proportion of mutants among thesurvivors.

Gene Exchange Mutagenesis of the purC Gene of M. tuberculosis

The selection protocol used for M. tuberculosis transposon mutagenesisalso seemed attractive for the positive selection of gene exchangemutants. Currently, none of the previously described strategies appearssuitable for mutagenesis of every gene in M. tuberculosis complexstrains (Norman et a;., 1995; Balasubramamian et al., 1996; Pelicic etal., FEMS Microbiol. Lett. 1996). The systems available are verydependent on the target gene, and have proved far less efficient forseveral “refractory” genes such as purC, which has previously failed tomutagenize in M. tuberculosis (unpublished data). Because M.tuberculosis purine auxotrophic mutants may have a vaccinal potential(Fields et al., 1986), the purC gene from the purine biosyntheticpathway was a perfect candidate for testing this new tool (Jackson etal., 1996). A mutated allele, purC::Km, was inserted into pPR27 alongwith xylE as a reporter gene. Since the gentamycin resistance gene isnot a reliable marker in M. tuberculosis, this reporter activity couldfacilitate the screening by discriminating possible allelic exchangemutants which have lost the xylE gene from SacB mutants with the wholevector integrated into the chromosome and which are thus phenotypicallyxylE⁺. Indeed, xylE expression in mycobacteria can easily be tested byspraying colonies on plates with a solution of catechol and observing abright yellow coloration (Curcic et al., 1994).

Plasmid p27CKX was introduced in M. tuberculosis by electroporation andtransformants were selected at 32° C. on 7H10-kanamycin. Severaltransformants were grown in liquid culture supplemented withhypoxanthin, a purine precursor. The culture was then plated at 39° C.on 7H10-kanamycin+2% sucrose+hypoxanthin plates. With an initialinoculum of 10⁷ colonies, 200 transformants were obtained oncounter-selective plates. All presented the expected phenotype forallelic exchange mutants: Suc^(r), Km^(r), XylE⁻. The phenotypicanalysis confirmed that they were purine auxotrophs, as they were notable to grow on Sauton medium, a synthetic medium containing no purinebases, without the addition of hypoxanthin. To unambiguously confirmthat the selected clones were allelic exchange mutants, several colonieswere grown m 7H9 supplemented with hypoxanthin. Genomic DNA wasextracted and analyzed by Southern-blotting using the purC gene as aprobe (FIG. 3). M. tuberculosis 103 DNA which was included as a control(WT), showed one hybridizing fragment of 2.5 kb. As expected for allelicexchange mutants, all the clones presented a single hybridizing fragmentapproximately 1.2 kb longer than that in the wild-type strain (FIG. 3).This 1.2 kb-increase corresponded to the size of the kanamycinresistance cassette which was inserted into the mutated allele.Therefore, all the tested transformants, selected on counter-selectiveplates, were indeed allelic exchange mutants. This confirmed thatts—SacB delivery vectors, in addition to being useful for transposonmutagenesis, are also highly efficient for gene exchange mutagenesis.

In vitro and in vivo Infection with the Wild Strains and the RecombinantStrains

1) In mice bone marrow macrophages

In contrast with the wild type Mt103 and BCG 1173P2 which multipliedinside macrophages between 0 and 15 days, (the wild type M. tuberculosislysed the macrophages before 4 days, which explains the cfu counts wereonly performed at 18h) the auxotrophic strains of M. tuberculosisMYC1551 and of BCG MYC 1552 did not multiply within the same period oftime. MYC 1552 was progressively eliminated from macrophages (FIG. 4).MYC1551 seemed to persist in the macrophages without multiplying as thenumber of cfu's recovered at day 15 was almost the same as at 18h (FIG.5). Moreover, the strain identified as pOMKC corresponding to MYC1551complemented with the wild type copy of the purC gene carried on aplasmid, behaved like the wild type Mt103. This result confirmed thatthe attenuation of MYC1551 is due to the inactivation of the purC geneand not to a polar effect of the mutation carried by this gene onadjacent genes.

2) In mice

As shown in FIGS. 7-9, strains MYC1551 and MYC1552 were attenuated ascompared to the wild type strains Mt103 and BCG 1173P2. MYC1552 did notmultiply at all (not even between day 1 and day 15) and wasprogressively eliminated from all organs. These results confirm what wasobserved in macrophages (FIG. 4). MYC1551 did not multiply on theoverall period between day 1 and day 20. Nevertheless, this strainseemed to persist in all organs during the same period of time. Theseresults also confirm what was observed in macrophages (FIG. 5).

EXAMPLE 2

Bacterial Strains and Culture Conditions

Escherichia coli XL1-Blue was used for cloning experiments and grown onliquid or solid Luria-Bertani medium. The mycobacterial strains, M.bovis BCG strain Pasteur 1173P2, M. tuberculosis MT103 (isolated from aTB patient) and H37Rv (ATCC 27294) were grown on liquid Middlebrook 7H9medium (Difco) supplemented with 0.05% Tween 80 or on solid Middlebrook7H10 or 7H11 medium (Difco). When required, kanamycin (20 μg/ml),gentamycin (10 μg/ml) or hypoxanthin (20 μg/ml), a purine precursor,were added to the growth media. M. tuberculosis H37Rv was obtained fromthe American Type Culture Collection, Rockville, Md., and stored as asingle-cell suspension at −70° C. (Grover et al., 1967).

Plasmid Construction

Plasmid pMJ104 was constructed by inserting the 3.7 kb BamBI fragmentcontaining purC::Km from pMJ101 into the BamHI site of pJQ200 (Pelicicet al., 1997). This plasmid is unable to replicate in mycobacteria. Theplasmid pMJ105 was created by excising the 1090 bp EcoRV—HincII fragmentfrom plasmid pMJ1, harbouring the purC gene flanked by 120 bp upstreamand 80 bp downstream, and by cloning it into plasmid pOMK (Jackson etal., 1996). pMJ105 is able to replicate in mycobacteria.

Experimental Animals

For these experiments, C57BL/6j or BALB/c female mice 6 to 8 weeks old(purchased from CERJ, Le Genest St Isle, France) were utilized. Theywere kept under good conventional housing conditions for up to 3 months.Mice inoculated with M. tuberculosis strains were maintained inbiohazard facilities and housed in cages contained within a safetyenclosure. Forty-seven male and female outbread Hartley-stin guinea pigs(Charles River Laboratories, Inc. Wilmington, Mass., USA) were utilizedin this study. They were individually housed in polycarbonate cages withstainless steel grid floors and feeders and were provided withcommercial guinea pig chow and tap water ad libitum. Each animal wasrandomly assigned a vaccine group and sacrifice interval. Prior tovaccination with the purC auxotrophic strain of M. tuberculosis and/orchallenge with virulent M. tuberculosis, the animals were moved into aBL3 biohazard suite and kept in individual stainless steel cages withgrid floors and water bottles.

Preparation and Infection of Mouse Bone Marrow Macrophages

Bone marrow cells were flushed from the femurs of 7 to 8 weeks-oldBALB/c mice and suspended in Dulbecco medium with low glucose (1g/liter) and high carbonate (3.7 g/liter) concentrations (Gibco BRL) andenriched with 10% heat-inactivated foetal calf serum (DominiqueDutscher), 10% L-cell conditioned medium and 2 mM glutamine. For theinfection assays, mouse bone marrow macrophages were seeded in 8-wellsLab-Tek chamber slides (Nalgen Nunc International) (5×10⁴ cells/well ina volume of 400 μl) and allowed to differentiate for 6 to 8 days. Analiquot of the M. tuberculosis suspensions used to infect macrophageswas plated onto Middlebrook 7H10 agar to establish the exact number ofbacteria in the inoculum. Prior to macrophage infection, mycobacteriawere washed twice in the cell culture medium described above, andsonicated in a sonicator bath (Branson 2200) for 15 seconds. Bacterialaggregates were allowed to sediment for 10 minutes. The top 500 μl wererecovered and bacterial concentrations were adjusted to 2×10⁴bacteria/ml with cell culture medium. The infection assay was asfollows. The culture medium of each Lab-Tek chamber slide well wasremoved and replenished with 500 μl of the mycobacterial suspensiondescribed above in order to have a multiplicity of infection (MOI) of1:1. Control wells containing non-infected macrophages received 500 μlfresh culture medium. Infected and non-infected cultures were incubatedat 37° C. in a 5% CO₂ atmosphere for 18 hours. After 18 hours, infectionwas terminated by removing the overlaying medium and washing each wellthree times with 500 μl Hank's buffered salt solution (HBSS) (Gibco BRL)before adding 400 μl of fresh culture medium per well. At day 1 (18 h),4, 7 and 11, the number of intracellular colony forming units (CFU) wasevaluated. For this, macrophages monolayers were washed three times inHBSS buffer and then lysed in 100 μl of cell culture lysis reagent(Promega). Different dilutions of this lysis solution were plated onto7H 10 or 7H 10 supplemented with hypoxanthin plates to perform themycobacterial colony counts. At each time point, a Lab-Tek chamber slidecontaining infected macrophages and cultivated in the same conditionswas subjected to staining for acid-fast bacilli in order to check themacrophages viability. This infection experiment was carried out induplicate.

Mycobacterial Multiplication in Mice

Mice were infected intravenously either with either 10⁵ cfu of MYC1551and MT103 or 10⁷ cfu of MYC1552 and BCG in 0.5 ml of phosphate salinebuffer. At every time point, mice were euthanized with CO₂, the spleen,lung and liver were removed aseptically and homogenized either manuallyusing two glass slides for the spleen and lung or using a Stomacher 80(Seward) homogenizer for the liver. Homogenates were resuspended in 10ml of buffer (Sauton 25% supplemented with 2% Middlebrook OADC (Difco),pH 7.5). For the lung, gentamycin were added at a final concentration of10 μg/ml to avoid contamination. Enumeration of bacteria in the organsof infected animals was performed by plating 10-fold serial dilutions(performed in Sauton 25%, pH 7.5) of organ homogenates on 7H11 medium(supplemented with hypoxanthin for the auxotroph mutants). Colonies werecounted after 3 to 4 weeks of incubation at 37° C. for the wild typestrains and after 6 to 8 weeks for the mutants. The data were expressedas the geometric means +/− standard deviation of counts obtained with 5to 6 mice.

Vaccination, Challenge and Necropsy of Guinea Pigs

Each guinea pig received approximately 10⁷ cfu of either wild type BCG,the corresponding BCG auxotrophic stain MYC1552, or the M. tuberculosisauxotrophic mutant MYC1551. A volume of 0.2 ml of vaccine or sterilephysiological saline (placebo) was injected subcutaneously into theinguinal region of each animal. Three, six and nine weekspost-vaccination, three animals from each of the three vaccine groupswere euthanized with a peritoneal injection of 2 ml of sodiumpentobarbital (Sleepaway; Fort Dodge Laboratories, Inc., Ft Dodge,Iowa). One-half of the spleen and the right lower lobe of the lung wereaseptically removed and homogenized separately in Teflon-glasshomogenizers in 4.5 ml of sterile physiological saline. The number ofviable mycobacteria in each organ was determined by inoculatingappropriate dilutions onto duplicate 7H10 plates supplemented withhypoxanthin. Data were expressed as mean log₁₀ number of viableorganisms per tissue. Nine weeks post vaccination, the challengeinoculum of H37Rv was rapidly thawed and diluted just prior toinfection. All animals were infected via the respiratory route by use ofan aerosol chamber as previously described (Wiegeshaus et al., 1970).The infecting inoculum of viable H37Rv was empirically adjusted toresult in the inhalation of 5 to 10 viable organisms per animal. Fiveweeks post-respiratory challenge, all remaining guinea pigs wereeuthanized by the intraperitoneal injection of 2 ml of sodiumpentobarbital. The abdominal and thoracic cavities were openedaseptically and the spleen and right lower lobe were removed forbacterial culture.

Lymphoproliferation Assay

Mitogen- and antigen-induced lymphoproliferation was assessed in vitroby an established procedure (Bartow and McMurray, 1989). Lymphocytesfrom the spleen were suspended in complete tissue culture medium andplated in 96 well microtiter plates (Falcon 3072, Becton Dickinson andCompany, Franklin Lakes, N.J.) at 2×10⁵ cells per well. Triplicatecultures were stimulated with purified protein derivative (PPD; StatensSeruminstitut, Copenhagen, DK) at concentrations of 25 μg/ml orconcanavalin A (Sigma) at a concentration of 10 μg/ml. Control culturesreceived cells and medium alone. The concacanavalin A (ConA) were usedas control as a non-specific inducer of lymphoproliferation. Thecultures were incubated for 4 days at 37° C. in a 5% CO2 environment;labeled with 1 μCi of tritiated thymidine per well for the last 6 hours,and harvested. The cellular uptake of thymidine was quantified in aliquid scintillation counter. The result were expressed as mean countsper minute (cpm) of stimulated cultures minus mean cpm of unstimulatedcells of the same source. The stimulation index (SI) was calculated bydividing the cpm in stimulated cultures by the cpm from unstimulatedcultures of the same animal's cells.

Tuberculin Skin Test

The delayed-type hypersensitivity reaction was evaluated by theintradermal injection of 0.1 ml of PPD containing 100 tuberculin unitsRT-23, Statens Seruminstitut) on a shaved area of the abdomen. The meandiameter of induration was measured in millimeters and recorded 24 hourslater.

Statistical Methods

Analysis of variance was utilized to test the effects of vaccination ontissue bacterial load. When significant treatment effects wereindicated, differences between means were assessed by Duncan's multiplerange test A 95% confidence level was set for all tests. All analyseswere performed using PC SAS 6.12 (SAS Institute, Cary, N.C.).

Construction of purC- Mutants of M. bovis BCG and M. tuberculosis

M. bovis BCG strain 1137P2 was electroporated using 2 μg of plasmidpMJ104. Two Km^(r) transformant were obtained. Their genomic DNA wereextracted and analysed by Southern blot using the purC gene as a probe.One of the two clones exhibited the correct hybridization pattern for anallelic exchange mutant The other one corresponded to the wild type andwas probably a Km^(r) mutant of M. bovis BCG. The candidate clone wasphenotypically tested. As expected, it required purines or hypoxanthin(a precursor of purine bases) to grow on 7H9 or 7H10. This mutant wasrenamed MYC1552.

The purC- mutant of M. tuberculosis MT103, named MYC1551, was obtainedas described in Pelicic et al., 1997. Like MYC1552, MYC1551 is dependenton the presence of purines or hypoxanthin in the medium to multiply.

Evaluation of the Reversion Frequency of MYC1551 and MYC1552

To estimate the stability of the mutation introduced by allelic exchangein M. bovis BCG and M. tuberculosis, cultures of MYC1551 and MYC1552grown in 7H9 containing kanamycin were plated on 7H10 with kanamycin andhypoxanthin or 7H10 alone. In all the experiments (repeatedindependently 4 and 2 times for MYC1552 and MYC1551 respectively), nocolony forming unit was detected on 7H10. In contrast, an average of8.10⁷ cfu/ml and 4.10⁸ cfu/ml were obtained respectively for MYC1551 andMYC1552 on 7H10 supplemented with hypoxanthin and kanamycin. Therefore,the reversion frequency was estimated to be lower than 10⁻⁸ events percell and per generation. The same experiment was repeated with culturesgrown in 7H9 liquid medium without kanamycin and the same results wereobtained.

Growth Characteristics of the Purine Auxotrophs in Macrophages

The ability of the purine auxotrophs to persist and multiply withinbone-marrow macrophages from C57BL/6 mice was evaluated. 5×10⁴macrophages were infected at a multiplicity of infection close to 1 withthe parental and the mutant strains. Over a period of 15 days, theinfection was followed by counting the viable bacteria (FIGS. 10A and10B). M. tuberculosis MT103 and M. bovis BCG 1137P2 multiplied withinthe macrophage with an apparent doubling time of 36 h and 59 hrespectively. At 8 days post-infection, macrophages were packed with M.tuberculosis and most of them lysed. With M. bovis BCG, the infectionwas followed for 15 days without apparent lysis of the macrophages. Thepurine auxotrophs exhibited totally different growth characteristics ascompared to their parental strains. In both cases, infections werecontained. MYC1551 persisted, but the number of viable bacteria did notincrease. MYC1552 was gradually eliminated and after 15 days less than3% of the inoculum was still viable. These results demonstrate that thedisruption of the purC gene alters the ability of M. tuberculosis and M.bovis BCG to multiply within mouse bone-marrow macrophages.

To confirm that this phenotype was due to the mutation of the purC geneand not to a polar effect of the kan gene insertion, a complementationexperiment was performed. Plasmid pMJ105 containing purC waselectro-transferred into MYC1551. Transformants no longer requiredpurines to grow in 7H9 or 7H10. MYC1551:pMJ105 was used to infectmacrophages (FIG. 10B). As expected, the presence of purC on the plasmidfully restored the ability of MYC1551 to multiply within macrophages.

Persistence of the Auxotrophic Mutants and Their Parental Couterparts inMice M. tuberculosis (10⁵ cfu) or M. bovis BCG (10⁶ cfu) were used toinfect BALB/c mice intravenously. The infection was followed by killingthe mice at different time points and counting the number of viablebacteria in the liver, spleen and lung (FIGS. 11 and 12).

For the wild type M. tuberculosis, there was a large increase in thenumber of cfu in the three organs during the first two or three weeks:2.5 logs in the spleen (day 14), 1 log in the liver (day 14), 3 logs inthe lung (day 21). At this time the infection was controlled; thebacillary load was stabilized both in the lung and the spleen at 10⁶ and10⁵ cfu per organ, respectively. In the liver, the bacterial burdendecreased to reach a plateau at 10⁴ cfu. For the purine auxotrophMYC1551, the situation was quite different. The initial multiplicationwas dramatically reduced with less than half a log of increase in everyorgan at the peak occurring at day 7. Then, the bacilli were graduallyeliminated from the three organs to become undetectable at day 63.

For BCG, the initial multiplication was slower than for M. tuberculosis:only half a log of increase in the spleen at day 14 and almost nomultiplication in the lung and the liver. From day 14 in both the liverand the spleen and from day 28 in the lung, the number of viable bacillidecreased gradually to reach 5×10³, 10⁴ and 5×10⁴ in the lung, liver andspleen, respectively, at day 70 when the experiment was stopped. Withthe BCG auxotroph (MYC1552), the number of bacilli began to decreaseimmediately after the infection, and no bacteria were recovered from thelung at day 42, or from the liver at day 56. In the spleen, a fewcolonies were recovered even at day 70 when the experiment was stopped.

These results established that the mutations in the purC gene of bothBCG and M. tuberculosis attenuated the virulence of these strains in themouse model. The experiment was repeated in a second susceptible mouseline, C57BL/6, with M. tuberculosis MT103 and MYC1551 but without anydifferences noted. MYC1551 and MYC1552 were eliminated very efficientlyto become almost undetectable at the end of the experiment (day 63 forM. tuberculosis and day 70 for BCG). However, they persisted for a whilesince bacteria were recovered from every organ during the first 6 weeks.The main difference in the behaviour of the two purine auxotrophs thusappears in the first week post infection. While MYC1551 increasedslightly in every organ tested, MYC1552 began to decline immediatelyafter the infection. After this short lag time, MYC1551 was cleared asefficiently as MYC1552.

Cell-mediated Immune Responses Induced by the Purine Auxotrophs andProtective Efficacy in the Guinea Pig Model

In order to evaluate the protective efficacy of the purine auxotrophs,the guinea pig was chosen because it is much more susceptible to M.tuberculosis infection than the mouse. Furthermore, BCG protection iseasier to demonstrate in the guinea pig. While a maximum of one logdifference in the cfu number can be obtained in the lung and spleen ofunimmunized as compared to BCG vaccinated mice, differences of one totwo logs in the lung and 4 logs in the spleen are often obtained betweennaive and BCG vaccinated guinea pigs following low-dose aerosolchallenge (McMurray, 1994).

Outbred Hartley strain guinea pigs were inoculated subcutaneously with10⁷ cfu of either BCG 1173P2, MYC1552 or MYC1551. Three animals fromeach treatment group were euthanized at three, six and nine weeks postinfection. T he number of viable mycobacteria was determined in one halfof the spleen and the right lower lobe of the lung. Three weekspost-infection, 870 cfu+/−651 of BCG 1173P2 were found in the spleens ofthree guinea pigs. In contrast, 90 cfu were recovered from only one ofthree guinea pigs infected with MYC1551, and no bacteria were found inany of the animals vaccinated with MYC1552. Six and nine weekspost-infection, no bacillus was recovered from the spleens of any animalof any of the three treatment groups. At no time point, were bacillirecovered from the lungs of any vaccinated animal. These results showthat the purine auxotrophs are attenuated even in the highly susceptibleguinea pig as well as they were in the mouse model.

The cell mediated immune response induced by the different strains wasevaluated by measuring the lymphocyte proliferation induced by PPD, andcutaneous delayed-type hypersensitivity. Nine weeks post-vaccination,all the vaccinated animals exhibited a detectable skin test reactionagainst tuberculin (Table 2). The mean induration diameters, measured 24hours following the intradermal injection, were 13.9+/−0.9, 15.5+/−1.0and 11.1+/−2.2 respectively for the BCG, MYC1552 and MYC1551 infectedguinea pigs. The lymphocyte proliferation to PPD was measured for thevaccinated animals euthanized six and nine weeks post vaccination (Table3). In every case, PPD induced a strong proliferative response whichdecreased between the sixth and ninth weeks post-immunization. Thestimulation indexes ranged from 4.4 to 8.9 at six weeks, and from 2.9 to6.1 at nine weeks and were not statistically different (p>0.05).

Nine weeks post-vaccination, the remaining animals were infected via theaerosol route with a dose empirically adjusted to result in theinhalation of 5 to 10 viable M. tuberculosis I37Rv bacilli per animal.Five weeks later, viable M. tuberculosis were recovered quantitativelyfrom the spleen and the lung (Table 4). In the lung, both MYC1551 andMYC1552 exhibited a level of protection comparable with the one obtainedwith BCG 1173P2, namely 1 log of difference in the cfu count between thevaccinated and unimmunized animals. As expected, vaccine-inducedprotection was most visible in the spleen. BCG and MYC1551 induced asignificant level of protection, while in guinea pigs vaccinated withMYC1552, the number of cfu were comparable to those observed in theunimmunized animals.

The Duncan Multiple Range Test indicated that there was no significantdifference in the number of tubercle bacilli recovered from the lungbetween different groups of vaccinated animals but that the differencebetween vaccinated and non-vaccinated groups was statisticallysignificant (p<0.05). In the spleen, analysis revealed that thedifference between BCG and MYC1551 was not significant. However, thenumber of mycobacteria in the spleens of animals vaccinated with BCG wassignificantly lower than the number in MYC1552-vaccinated andunimmunized controls.

TABLE 2 Tuberculin skin test on guinea pigs injected via the intradermalroute with 100 tuberculin units nine weeks post-infection. BCG MYC1552MYC1551 Control induration 13.0 16.0 5.0 0 diameter 12.5 17.0 12.0 0(mm) 17.5 13.5 18.5 0 13.5 11.0 0 13.0 9.0 0 Mean +/− SEM. 13.9 +/− 0.915.5 +/− 1.0 11.1 +/− 2.2 0

TABLE 3 Proliferation of lymphocyte from vaccinated guinea pigs to PPD.ConA (10 μg/ml) PPD (25 μg/ml) Group Necropsy Net cpm SI Net cpm SI BCG6 15784 41.4 4346 12.1 6 15502 6.4 3119 2.1 6 13494 8.8 1413 1.8 Mean+/− SEM 6 14927 +/− 721 18.9 +/− 11.3 2959 +/− 850 5.3 +/− 3.4 MYC1552 64381 11.1 8775 21.3 6 59445 16.6 1431 0.6 6 6336 4.4 2460 2.3 Mean +/−SEM 6 23387 +/− 18037 10.7 +/− 3.5 3268 +/− 2974 8.1 +/− 6.6 MYC1551 617321 13.3 400 1.3 6 4750 10.9 677 2.4 6 63885 143.0 3811 9.5 Mean+/−SEM 6 28652 +/− 17986 55.7 +/− 43.6 1629 +/− 1093 4.4 +/− 2.54 BCG 936372 149.9 1125 5.6 9 50064 186.9 1519 6.6 Mean +/− SD 9 43218 +/− 6846168.4 +/− 18.4 1322 +/− 197 6.12 +/− 0.5 MYC1552 9 14150 94.1 293 2.9MYC1551 9 25347 135.6 397 3.1 9 33211 124.3 796 4.0 9 50512 234.1 4883.3 Mean +/− SEM 9 36357 +/− 7432 164.7 +/− 34.9 560 +/− 121 3.4 +/− 0.2

TABLE 4 Protective efficacy of the different vaccine strains againstlow-dose pulmonary challenge in guinea pigs. Lung Spleen (Log10 (Log10Group Viable Mycobacteria) Viable Mycobacteria) BCG 4.9 4.1 3.8 2.8 4.30 5.1 0 4.2 0 Mean +/− SEM 4.5 +/− 0.2 1.4 +/− 0.9 MYC1552 4.7 4.7 5.04.0 5.0 Mean +/− SEM 4.8 +/− 0.2 4.6 +/− 0.3 MYC1551 5.1 4.3 5.3 4.0 2.34.6 3.4 5.1 1.3 Mean +/− SEM 4.6 +/− 0.2 3.1 +/− 0.9 unimmunizedcontrols 5.3 5.9 5.2 4.8 5.4 4.7 6.0 Mean +/− SEM 5.6 +/− 0.2 4.7 +/−0.1

Thus, with the goal of developing a novel vaccine against tuberculosis,we constructed and evaluated the attenuation and protective efficacy ofpurC- auxotrophic mutant strains of M. tuberculosis and M. bovis BCGcarrying a defect in their purine biosynthetic pathway. This approachwas justified by the fact that attenuated strains are generally morepotent than non-living vaccines in stimulating cell-mediated immuneresponses which are effective against intracellular pathogens (Brown etal., 1993), and because, in theory, they produce most of the antigensnormally expressed in vivo by the pathogens. Thus, immune responses arestimulated in ways which closely resemble those detected during normalinfection. Moreover, by constructing an auxotrophic strain derived fromM. tuberculosis, one would derive a vaccine candidate antigenicallyidentical to the pathogen against which protection was desired.

As shown for other purine auxotrophs of intracellular pathogens whichreside inside vacuoles (McFarland & Stocker, 1987; O'Callaghan et al.,1988; Crawford et al., 1996), M. tuberculosis (MYC1551) and M. bovis BCG(MYC1552) purine deficient mutants were attenuated both in an in vitromacrophage model and in vivo in mice and guinea pigs. Since theintroduction of the purC gene on a plasmid into the M. tuberculosisauxotroph fully restores its ability to replicate and lyse themacrophages, one can conclude that restriction for growth inside thesecells is due to the insertional disruption of the purC gene alone andnot to polarity affecting the expression of adjacent genes.

In contrast to what was observed with their respective parental strains,the number of MYC1551 in mouse bone-marrow macrophages did not increaseover a 15 days period of time, but remained approximately constant,whereas the number of MYC1552 steadily decreased from the first daypost-infection. In the absence of purine bases in the medium, mostlikely resembling the situation within the phagosomal compartment, bothauxotrophs were unable to multiply. Thus, this difference inintracellular persistence between MYC1551 and MYC1552 probably reflectsthe different abilities of these M. tuberculosis and M. bovis BCGderivatives to resist phagocytic cells defenses and/or to make thephagosome in which they reside more hospitable.

The behavior of the auxotrophs in mice reflects the results obtained inisolated macrophages: the MYC1551 strain was progressively eliminatedafter a lag period of two to three weeks during which the number of cfuin all organs remained almost constant, whereas elimination of MYC1552began immediately after the infection. Overall, both strains wereeliminated at about the same rate from mice. In guinea pigs, fewerMYC1551 than BCG and no MYC1552 were found in the spleens three weekspost-immunization. Finally, based on data obtained from the macrophagesand guinea pigs experiments in which the same infecting doses were usedfor all strains, it seems that both MYC1551 and MYC1552 are moreattenuated than BCG.

Protection studies demonstrated that all three strains (BCG, MYC1551 andMYC1552) had equal statistically significant protective effects againstan aerosol challenge with virulent M. tuberculosis H37Rv, as assessed bythe reduction in cfu in the lungs of vaccinated guinea pigs. In thespleen, where a wider “protective window” is obtained, MYC1551 showed asignificant protective effect as compared to unimmunized controls. Thisprotective effect of MYC1551 appeared slightly less efficient than thatconferred by BCG, although the difference was not statisticallysignificant due to the heterogeneity of the guinea pigs responses toinfection in every vaccination group. In the same organ, MYC1552 seemedto induce no protective response at all.

All three strains elicited non-statistically different strong DTHresponses nine weeks post-infection, and stimulated PPD-inducedlymphoproliferative responses six and nine weeks post-infection (withBCG the most potent in this respect after nine weeks), suggesting thatboth auxotrophs are able to induce cell-mediated immune responses ofabout equal intensity as those induced by the BCG vaccine. Therefore,the differences in the protective efficacies of BCG, MYC1551 and MYC1552may have resulted from differences in the “quality” of the cellularimmune responses they induce, rather than in the intensity. In fact, itis probable that an attenuated strain of mycobacteria needs to retain alimited ability to multiply in host cells and disseminate and persistwithin the host in order to be able to induce protective immuneresponses. This idea was originally pointed out by Kanai's work (1966),in which the protective efficacy of a streptomycin-dependent strain ofM. tuberculosis was evaluated in guinea pigs and mice. In this system,streptomycin was used to induce the multiplication of the immunizingstrain prior to challenge with virulent H37Rv tubercle bacilli.Experiments showed that only the M. tuberculosis that had multipliedduring the immunization period conferred protection to the animals, withnon-multiplying bacteria displaying poor protection.

Thus, the way an attenuated strain establishes an infection in the host,probably more than persistence in itself, seems to be important forinducing protective immune responses. Similar conclusions were drawn byO'Callaghan and collaborators (1988), in which the protective efficaciesof a purA and of an aroA Salmonella typhimurium mutant, respectivelydeficient in the synthesis of adenine and aromatic amino-acids, werecompared in relation to the infection they established in mice. Theseobservations highlight the difficulty encountered when using livevaccines to reach the right balance between attenuation andimmunogenicity, since over-attenuated bacteria may not produce in vivosome key antigens necessary for the induction of a protective immunity.The physiological state of the mutant bacilli might also influence theway their antigens are processed inside the macrophage and, thus,presented to the T lymphocytes.

The present work provides evidence that rationally attenuated strains ofM. tuberculosis can protect guinea pigs against pulmonary tuberculosis.Finding an attenuated strain of the virulent tubercle bacillus whichwill have a greater efficacy than BCG seems reasonable if one considersthat M. tuberculosis expresses additional highly immunogenic antigenssuch as ESAT 6 which the BCG Jacks (Mahairas et al., 1996). However,this finding also seems dependent upon the level of attenuation of thevaccine candidate. Inactivating virulence genes instead of, or inaddition to, “house-keeping” genes might be necessary to obtain a M.tuberculosis mutant which will multiply at a similar rate as BCG withthe same level of attenuation.

In summary, a single, very simple system which can be used for easymutagenesis of M. tuberculosis either by allelic exchange was designed.Using purC, a gene that was previously unable to mutate in M.tuberculosis, it was demonstrated that ts—SacB vectors can also be usedfor allelic exchange mutagenesis. All the clones obtained after adouble-selection were indeed allelic exchange mutants. The M.tuberculosis purine auxotrophic mutant presents vaccmal potential(Fields, et al., 1986) and may also be used for the development of anIVET technology allowing the selection of mycobacterial genespreferentially expressed in vivo (Mahan et al., 1993; incorporatedherein by reference). Provided that its function is dispensable to thecell, it is reasonable to assume that the same protocol should allowmutagenesis of virtually every gene of M. tuberculosis through geneexchange.

This new tool should greatly contribute to the genetic analysis of M.tuberculosis pathogenicity following Koch's postulates: it allowscreation of deemed mycobacterial mutants by allelic exchange which waspreviously difficult, or even unfeasible. It opens the way not only tostudying the roles in pathogenicity of defined mycobacterial genes whichmay or not present similarities to known virulence factors from otherbacterial pathogens, but also to the rational construction of attenuatedstrains which could be more effective than the BCG as antituberculousvaccines.

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We claim:
 1. The recombinant mycobacterium strain MYC 1551 (C.N.C.M. No.I-1871).
 2. The recombinant mycobacterium strain MYC 1552 (C.N.C.M. No.I-1872).
 3. A recombinant mycobacterium strain of pathogenic origincapable of replicating in a macrophage of a host, said strain containingin its chromosome or on a plasmid a counterpart to a gene in the wildtype mycobacterium coding for a protein necessary for the biosynthesisof a purine or a pyrimidine base, wherein said counterpart gene in therecombinant mycobacterium has been inactivated by at least one pointmutation or by addition, deletion, or substitution of one or more basepairs, wherein said mycobacterium strain is selected from the groupconsisting of M. Bovis-BCG, M. tuberculosis, and M. smegmatis, and saidinactivated gene is a purC or purL gene.